In a previous report, we demonstrated that glutathione (GSH) played an important role in cadmium (Cd) detoxification in Rhizobium leguminosarum cells, but the molecular role of this tripeptide remained to be elucidated. In this work, we report an efficient extraction of GSH-Cd complexes in two R. leguminosarum strains with different levels of tolerance to Cd, through a modified protocol, originally developed for metal-phytochelatins complexes. Rhizobium cells were grown under the presence of 1 mM Cd (tolerant strain) and 0.25 mM Cd (sensitive strain). Cells were harvested and different sub-cellular Cd fractions were extracted: loosely bound Cd was extracted with water, in an ultrasonic bath; intracellular Cd was sequentially extracted with HEPES buffer and wall-bound Cd was extracted with acid. Intracellular Cd was separated through size exclusion chromatography, and the amount of Cd in collected fractions was analysed. Peptide peaks containing the higher Cd concentrations were analysed by RP-HPLC analysis with pre-column derivatisation. In both strains, the same percentage of Cd was retained in cell walls, hence demonstrating an effective avoidance mechanism, but that was not responsible for the differences in tolerance, displayed by the two strains. Intracellular Cd accumulation was higher in the tolerant strain and metal ions were mainly chelated by small-weight peptides. HPLC analysis revealed that GSH was the main Cd chelator in Rhizobium, being responsible for sequestering 75% of intracellular Cd in the tolerant strain. The sensitive strain presented a less effective Cd complexation, only 23% of intracellular Cd was sequestered by GSH. Our findings add a novel and important aspect to the proposed role of GSH in heavy metal coping for bacteria, in general. These results can be useful in developing biotechnological strategies for Cd bioremediation procedures and open novel perspectives for the improvement of metal tolerance in soil bacteria. (c) 2006 Elsevier Inc. All rights reserved.